Numerical simulation of DNA sample preconcentration in microdevice electrophoresis

A numerical model is presented for the accurate and efficient prediction of preconcentration and transport of DNA during sample introduction and injection in microcapillary electrophoresis. The model incorporates conservation laws for the different buffer ions, salt ions, and DNA sample, coupled through a Gaussian electric field to account for the field modifications that cause electromigration. The accuracy and efficiency required to capture the physics associated with such a complex transient problem are realized by the use of the finite element-flux corrected transport (FE-FCT) algorithm in two dimensions. The model has been employed for the prediction of DNA sample preconcentration and transport during electrophoresis in a double-T injector microdevice. To test its validity, the numerical results have been compared with the corresponding experimental data under similar conditions, and excellent agreement has been found. Finally, detailed results from a simulation of DNA sample preconcentration in electrophoretic microdevices are presented using as parameters the electric field strength and the other species concentrations. The effect of the Tris concentration on sample stacking is also investigated. These results demonstrate the great potential offered by the model for future optimization of such microchip devices with respect to significantly enhanced speed and resolution of sample separation.     

Recent advances in DNA sequencing by capillary and microdevice electrophoresis

A number of significant improvements in the electrophoretic performance and design of DNA sequencing devices have culminated in the introduction of truly industrial grade production scale instruments. These instruments have been the workhorses behind the massive increase in genomic sequencing data available in public and private databases. We highlight the recent progress in aspects of capillary electrophoresis (CE) that has enabled these achievements. In addition, we summarize recent developments in the use of microfabricated devices for DNA sequencing that promise to bring the next leap in productivity.     

Recent developments in DNA sequencing by capillary and microdevice electrophoresis

The present review covers papers published in the years 1997 and 1998 on DNA sequencing by capillary and microdevice electrophoresis. The article does not include other electrophoretic DNA applications such as analysis of oligonucleotides, genotyping, and mutational analysis. Capillary gel electrophoresis (CGE) is starting to become a viable competitor to slab gel electrophoresis for DNA sequencing. Commercially available multicapillary array sequencers are now entering sequencing facilities which to date have totally relied on traditional slab gel technology. CGE research on DNA sequencing therefore becomes increasingly concerned with the critical task of fine-tuning the operational parameters to create robust sequencing systems. Electrophoretic microdevices are being considered the next technological step in DNA sequencing by electrophoresis.     

Capillary electrophoresis of biological particles: viruses, bacteria, and eukaryotic cells

A review about the application of electrophoretic methods in the capillary format for the investigation of large biological assemblies like viruses, bacteria, yeast or entire mammalian cells is given. These entities are of a size ranging between some nanometers and tens of micrometers. They can form colloidal solutions or dispersions and move under the influence of an electric field. They are separated by zone electrophoresis according to their different electrophoretic velocity, and characterized by the electrophoretic mobility, which is easily determinable in free solution in capillaries or in other microdevices. As the charge of these particles, when being amphoteric, is pH-dependent, isoelectric focusing can also be carried out and the capillary format is increasingly being employed for their separation and determination of pI values. Furthermore, interactions with ligands can be assessed by various modes of affinity capillary electrophoresis. Capillary zone electrophoresis has thus become a valuable tool for investigation of large macromolecular assemblies in the field of biochemistry, clinical chemistry, toxicology, and nutrition chemistry amongst many others.     

Chiral separation of deprenyl-N-oxide isomers by capillary electrophoresis using various cyclodextrin derivatives

Chiral separation of deprenyl-N-oxide isomers is presented using capillary electrophoresis in the presence of various cyclodextrin (CD) derivatives. This recently identified metabolite of R-(-)-deprenyl may possess desirable pharmacological activities. The effect of the cavity size and the substituents of the CD are examined on the enantiomer resolution of the compound having an asymmetric center on a heteroatom. The importance of hydrophilic or hydrogen bonding interaction, as well as the position of the interacting groups is demonstrated. Outstanding selectivity and resolution values are achieved using the chargeable carboxymethyl-beta-CD. 2-Hydroxypropyl-beta-CD is also suitable for the enantiomer separation of the analyte. Native beta-CD and carboxyethyl-beta-CD provide only poor enantioselectivity, whereas heptakis-(2,6-di-O-methyl)-beta-CD is capable of separating only the diastereomers. No chiral resolution can be observed in the presence of gamma-CD.     

Recent advances in capillary electrophoresis of proteins, peptides and amino acids

The current status of high-performance capillary electrophoresis as an analytical separation method for proteins, peptides and amino acids is assessed. Recent advances in suppressing the effects of electroosmotic flow and irreversible adsorption of proteins at the capillary wall are reviewed, together with procedures for optimal separations of peptides and amino acids. The detection aspects emphasize the role of laser-induced fluorescence and capillary electrophoresis/mass spectrometry in high-sensitivity measurements.     

Separation of basic proteins and peptides by capillary electrophoresis using a cationic surfactant

Incorporation of a low concentration of cetyltrimethylammonium bromide (CTAB) in the running electrolyte is shown to dynamically coat the silica capillary and to reverse the direction of electroosmotic flow. The CTAB coating prevented interaction of proteins with the capillary surface and enabled sharp peaks to be obtained in the electropherograms. A systematic study of experimental parameters demonstrated the importance of selecting a suitable buffer electrolyte and an appropriate pH. Excellent separations were obtained for five proteins, three enkephalins, and six dipeptides with an efficiency of approximately 500,000 theoretical plates per meter. The method developed is very simple to perform and was found to give excellent reproducibility.     

High-throughput separation of DNA and proteins by three-dimensional geometry gel electrophoresis: feasibility studies

We report a novel separation method that is applicable to both DNA and protein samples, based on electrophoresis in a three-dimensional (3-D) geometry. In contrast to conventional electrophoresis, samples are applied in a two-dimensional, planar array to one of the surfaces of a 3-D geometry separation medium. Loading onto a plane results in a very high sample capacity. Sample migration and separation occur along the third spatial dimension, which is perpendicular to the loading plane. The key problem of electrophoresis in a 3-D geometry separation setup is that temperature gradients are caused by Joule's heat, affecting the electrical conductivity and viscosity of the separation medium. A means of achieving straight sample migration under these circumstances is to force heat flow through the separation medium parallel to the axis of sample migration. This can be done by dissipating the heat via the electrode sides of the gel and blocking any other heat transfer. The separation of DNA and proteins by this method has been tested using agarose gel electrophoresis, polyacrylamide gel electrophoresis, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Data were acquired off-line by conventional staining methods as well as on-line by detection of laser-induced fluorescence. We describe how to excise samples from the separation medium for preparative purposes. Possible unique applications of this 3-D geometry electrophoresis separation method are also discussed.     

Human genomic DNA analysis using a semi-automated sample preparation, amplification, and electrophoresis separation platform

The growing importance of analyzing the human genome to detect hereditary and infectious diseases associated with specific DNA sequences has motivated us to develop automated devices to integrate sample preparation, real-time PCR, and microchannel electrophoresis (MCE). In this report, we present results from an optimized compact system capable of processing a raw sample of blood, extracting the DNA, and performing a multiplexed PCR reaction. Finally, an innovative electrophoretic separation was performed on the post-PCR products using a unique MCE system. The sample preparation system extracted and lysed white blood cells (WBC) from whole blood, producing DNA of sufficient quantity and quality for a polymerase chain reaction (PCR). Separation of multiple amplicons was achieved in a microfabricated channel 30 microm x 100 microm in cross section and 85 mm in length filled with a replaceable methyl cellulose matrix operated under denaturing conditions at 50 degrees C. By incorporating fluorescent-labeled primers in the PCR, the amplicons were identified by a two-color (multiplexed) fluorescence detection system. Two base-pair resolution of single-stranded DNA (PCR products) was achieved. We believe that this integrated system provides a unique solution for DNA analysis.     

Enantiomer separation of drugs by capillary electrophoresis using proteins as chiral selectors

The separation of drug enantiomers using proteins as the chiral selectors in capillary electrophoresis (CE) is considered in this review. The proteins used include albumins such as bovine serum albumin, human serum albumin and serum albumins from other species, glycoproteins such as alpha1-acid glycoprotein, crude ovomucoid, ovoglycoprotein, avidin and riboflavin binding protein, enzymes such as fungal cellulase, cellobiohydrolase I, pepsin and lysozyme and other proteins such as casein, human serum transferrin and ovotransferrin. Protein-based CE is carried out in two modes: in one proteins are immobilized or adsorbed within the capillary, or protein-immobilized silica gels are packed into the capillary (affinity capillary electrochromatography mode), and in the other proteins are dissolved in the running buffer (affinity CE mode). Furthermore, the advantages and limitations of the two modes and the factors affecting the chiral separations of various drugs by protein-based CE are discussed.     

Analytical and micropreparative separation of peptides by capillary zone electrophoresis using discontinuous buffer systems.

The tiny injection volumes that are usually necessary to maintain the high efficiency of capillary zone electrophoresis present a major problem if only limited sample amounts are available. To increase the sample load, discontinuous buffer systems were developed that allow the on-column concentration of dilute samples. Injection volumes can be increased in this way by at least a factor of 30. These stacking systems were applied to the analysis of tryptic peptides, to the purity checking of high-performance liquid chromatographic fractions and for the micropreparative separation of peptides with subsequent amino acid sequence analysis.     

Molecular imprinting of peptides and proteins in aqueous media

Molecular imprinting has received significant attention in recent years, as it provides a viable method for creating synthetic receptors capable of selectively recognizing specific target molecules. Despite significant growth within the field, the majority of template molecules studied thus far have been characterized by their low molecular weight and insolubility in aqueous systems. In biological systems, molecular recognition events occur in aqueous media. Therefore, in order to create molecularly imprinted polymers capable of mimicking biological processes, it is necessary to synthesize artificial receptors which can selectively recognize their respective target biological macromolecules such as peptides and proteins in aqueous media. In this review, we discuss the challenges associated with the imprinting of peptides and proteins in aqueous media. In addition, we discuss the significant progress which has been made within the field.     

Chiral separation of amino acids and peptides by capillary electrophoresis

Chiral separation of amino acids and peptides by capillary electrophoresis (CE) is reviewed regarding the separation principles of different approaches, advantages and limitations, chiral recognition mechanisms and applications. The direct approach details various chiral selectors with an emphasis on cyclodextrins and their derivatives, antibiotics and chiral surfactants as the chiral selectors. The indirect approach deals with various chiral reagents applied for diastereomer formation and types of separation media such as micelles and polymeric pseudo-stationary phases. Many derivatization reagents used for high sensitivity detection of amino acids and peptides are also discussed and their characteristics are summarized in tables. A large number of relevant examples is presented illustrating the current status of enantiomeric and diastereomeric separation of amino acids and peptides. Strategies to enhance the selectivity and optimize separation parameters by the application of experimental designs are described. The reversal of enantiomeric elution order and the effects of organic modifiers on the selectivity are illustrated in both direct and indirect methods. Some applications of chiral amino acid and peptide analysis, in particular, regarding the determination of trace enantiomeric impurities, are given. This review selects more than 200 articles published between 1988 and 1999.     

Laser-induced fluorescence detection schemes for the analysis of proteins and peptides using capillary electrophoresis

Over the past few years, a large number of studies have been prepared that describe the analysis of peptides and proteins using capillary electrophoresis (CE) and laser-induced fluorescence (LIF). These studies have focused on two general goals: (i) development of automatic, selective and quick separation and detection of mixtures of peptides or proteins; (ii) generation of new methods of quantitation for very low concentrations (nm and subnanomolar) of peptides. These two goals are attained with the use of covalent labelling reactions using a variety of dyes that can be readily excited by the radiation from a commonly available laser or via the use of noncovalent labelling (immunoassay using a labelled antibody or antigen or noncovalent dye interactions). In this review article, we summarize the works which were performed for protein and peptide analysis via CE-LIF.     

Recent advances in microchip electrophoresis for analysis of pathogenic bacteria and viruses

Life-threatening diseases, such as hepatitis B, pneumonia, tuberculosis, and COVID-19, are widespread due to pathogenic bacteria and viruses. Therefore, the development of highly sensitive, rapid, portable, cost-effective, and selective methods for the analysis of such microorganisms is a great challenge. Microchip electrophoresis (ME) has been widely used in recent years for the analysis of bacterial and viral pathogens in biological and environmental samples owing to its portability, simplicity, cost-effectiveness, and rapid analysis. However, microbial enrichment and purification are critical steps for accurate and sensitive analysis of pathogenic bacteria and viruses in complex matrices. Therefore, we first discussed the advances in the sample preparation technologies associated with the accurate analysis of such microorganisms, especially the on-chip microfluidic-based sample preparations such as dielectrophoresis and microfluidic membrane filtration. Thereafter, we focused on the recent advances in the lab-on-a-chip electrophoretic analysis of pathogenic bacteria and viruses in different complex matrices. As the microbial analysis is mainly based on the analysis of nucleic acid of the microorganism, the integration of nucleic acid-based amplification techniques such as polymerase chain reaction (PCR), quantitative PCR, and multiplex PCR with ME will result in an accurate and sensitive analysis of microbial pathogens. Such analyses are very important for the point-of-care diagnosis of various infectious diseases.     

Molded separation media: An inexpensive,efficient, and versatile alternative to packed columns for the fast HPLC separation of peptides,proteins, and synthetic oligomers and polymers

A simple molding process carried out within the confines of a chromatographic column has been used for the preparation of macroporous poly(glycidyl methacrylate-co-ethylene dimethacrylate) and poly(styrene-co-divinylbenzene) rods. The novel monolithic separation media that are obtained are useful for the HPLC separation of biological and synthetic polymers. The presence of large pores with a diameter of about 1 μm makes the molded rod columns easily permeable to eluents. Therefore, the back pressure of these columns is modest even at high flow rates. In contrast to the conventional HPLC columns packed with beads, all of the mobile phase flows through the continuous monolithic medium. As a result of this total convection, the efficiency of the molded media is almost independent of the flow rate. This improves significantly the separation ability of the rod columns and very fast separations of macromolecules such as peptides, proteins, and synthetic polymers have been demonstrated.     

Electrokinetic separation of peptides and proteins using a polyvinylamine-coated capillary with UV and ESI-MS detection

In order to accomplish the analysis of peptides and proteins by capillary electrophoresis, Lupamin, a high-molecular-weight linear polyvinylamine (PVAm) polymer, was introduced to modify the inner wall of fused-silica capillaries by physical absorption. Thanks to the high density of positively charged amino groups in Lupamin under acidic conditions, not only is a strong reversed electroosmotic flow generated in the coated capillary but the adsorption of analytes on the inner wall of the capillary is also efficiently eliminated. It has been demonstrated that the Lupamin-coated capillary can be used to advantage for the rapid analysis of amino acids, peptides, and proteins with good resolution and peak shape by capillary electrophoresis. In order to evaluate the basic feature of a Lupamin-coated capillary, electroosmotic flows generated by a Lupamin coating layer under different conditions including pH, coating time, concentration, and the composition of electrolytes on Lupamin-coated and uncoated capillaries were investigated. Furthermore, electrospray ionization-mass spectrometry (ESI-MS) detection was carried out for the analysis of amino acids and peptides.     

Rapid separation of four probiotic bacteria in mixed samples using microchip electrophoresis with laser-induced fluorescence detection

Microchip electrophoresis (MCE) coupled to laser-induced fluorescence detection was applied to the rapid separation of Bifidobacterium, Lactobacillus casei, Lactobacillus acidophilus, and Enterococccus faecalis. All bacteria were quickly separated within 150?s using a running buffer of pH 8.5 containing Tris, borate, EDTA, and poly(ethylene oxide). The latter was crucial to reduce the bacterial adsorption on the walls of the microchannels. The pH of 8.5 warrants that bacteria carry a negative charge at their surface and thus display good electrophoretic performance. The method was used to analyze medical samples containing these probiotics, and the results showed that the identification and detection of bacteria by MCE is advantageous in terms of sample consumption, waste production, time of analysis, and instrumental effort.

Separation of peptides and proteins by capillary electrophoresis using acidic buffers containing tetraalkylammonium cations and cyclodextrins

A method for improving separations of peptides and other positively charged species in capillary zone electrophoresis with untreated capillaries using acidic buffers containing tetraalkylammonium cations is described. Tetramethylammonium and tetrabutylammonium cations dynamically modify the capillary surface, leading to a reversal in the direction of the electroosmotic flow. As a result, the adsorption of positively charged peptides and proteins is minimized, and resolution and peak capacity are improved as the migration of cationic analytes is counterbalanced by the electroosmotic flow. The combining effect of reversing electroosmotic flow and cyclodextrin inclusion complexation on separations of closely related peptides and a protein mixture, as well as tryptic digest of hemoglobin is demonstrated.